CUT&Tag-IT^® Express Assay Kit

The CUT&Tag-IT Express Assay Kit delivers a faster, more streamlined workflow than the original CUT&Tag-IT Assay Kits (Cat. Nos. 53160 and 53165), designed for efficiency, scalability, and ease of use.

The Express format uses 0.2 mL tubes instead of 1.5 mL tubes, enabling shorter wash steps and full compatibility with multi-channel pipetting for higher-throughput processing. It also features silica bead–based DNA purification in place of column purification, significantly reducing hands-on time.

To further simplify experimental setup, the Express Kit includes both anti-mouse and anti-rabbit secondary antibodies in a single kit—eliminating the need for separate kits based on primary antibody species.

• Tube format: 0.2 mL (Express) vs. 1.5 mL (Original)
• Workflow speed: Faster wash steps and fewer transfers
• Purification method: Silica beads (Express) vs. spin columns (Original)
• Throughput: Multichannel pipette compatible
• Target Antibody Compatibility: mouse and rabbit secondaries included (Express) vs separate anti-mouse and anti-rabbit kits (Original)

Related product: Original CUT&Tag-IT Assay Kits (Cat. No. 53160 and 53165)

CUT&Tag is a native, antibody-guided tagmentation method that profiles chromatin binding sites without fixation, sonication, or immunoprecipitation. Compared to ChIP-seq, CUT&Tag requires fewer cells, produces lower background, and generates sequencing-ready libraries in a single step.

• No crosslinking: Performed in native cells
• No sonication: Chromatin is cleaved by tethered pA-Tn5
• Lower input: Works with fewer starting cells
• Integrated library prep: Tagmentation generates sequencing-ready DNA
• Lower background: Higher signal-to-noise than ChIP-seq
• Lower sequencing depth: Fewer reads needed for robust results

These advantages make CUT&Tag-IT ideal for limited samples, rapid workflows, and cost-effective chromatin profiling.

Choose CUT&Tag when you have limited starting material and want high-resolution, low-background profiling of histone modifications or selected chromatin-associated proteins. CUT&Tag-IT is especially well suited for native cells, rapid workflows, and low sequencing depth.

• Best for: Histone marks and chromatin features
• Sample input: Low cell numbers
• Signal quality: High signal-to-noise, low background
• Workflow: Fast, integrated library preparation

For comprehensive analysis of genome-wide transcription factor binding—particularly for difficult or low-abundance targets—traditional ChIP-seq may still be preferred.

Yes. The CUT&Tag-IT^® Assay Kit includes all core reagents required to perform CUT&Tag, including the pre-assembled protein A–Tn5 (pA-Tn5) transposomes loaded with sequencing adapters.

• Included: pA-Tn5 transposomes, buffers, and reaction components
• Not included: Standard laboratory equipment and general reagents
• Documentation: A complete list of additional materials is provided in the kit manual

The only items not supplied are standard lab consumables and instruments, which are listed under Additional Materials Required in the CUT&Tag-IT^® Express Assay Kit manual.

No. The standard Active Motif ChIP-Seq Spike-In Normalization method is not compatible with the CUT&Tag-IT^® Assay Kit.

CUT&Tag-IT uses a targeted, antibody-tethered tagmentation approach that does not support external spike-in controls designed for ChIP-Seq workflows.

No. CUT&Tag-IT does not require an INPUT control.

Unlike ChIP-Seq, CUT&Tag uses a tethered pA-Tn5 transposase that targets DNA only at antibody-bound sites. This targeted mechanism eliminates the need for an INPUT control to normalize background signal.

The recommended input for the CUT&Tag-IT^® Express Assay Kit is 50,000 to 100,000 cells per reaction. Useful CUT&Tag data can also be generated from as few as 5,000 cells, depending on target abundance and antibody performance.

• Recommended range: 50,000 – 100,000 cells
• Minimum demonstrated input: ~5,000 cells
• Performance depends on: Target abundance and antibody quality

Lower input experiments may require careful optimization and appropriate positive controls to ensure robust results.

Prepare cells exactly as described in the CUT&Tag-IT^® manual. Cells must remain unfixed and viable.

For adherent cells, do not use trypsin, as it damages the surface epitopes required for binding to Concanavalin A beads.

• Use enzyme-free dissociation
• Scrape cells or use a rubber policeman
• Avoid harsh mechanical or enzymatic treatments

Yes—if the cells were cryopreserved. Flash-frozen cell pellets are not compatible with the CUT&Tag-IT workflow.

• Use cryopreserved cells only
• Do not use flash-frozen pellets
• Thaw gently and handle as described in the protocol

To cryopreserve cells correctly, follow Active Motif’s Services sample preparation protocol.

Concanavalin A (Con A) beads should appear evenly suspended and uniformly opaque. When cells bind, the beads become visibly larger, darker, and form soft aggregates that move together in the tube.

• Unbound beads: fine, evenly dispersed particles
• Cell-bound beads: larger clusters that settle more quickly
• Clumping or debris: indicates poor cell prep or bead handling

Watch the short video below for a visual reference of proper bead appearance and binding:

Use H3K27me3 as the technical positive control and a no-primary-antibody sample as the negative control.

• Positive control: H3K27me3 antibody (Cat. No. 39157) — confirms reagent performance and workflow integrity
• Negative control: Secondary antibody only (no primary) — measures background from pA-Tn5 and the secondary antibody

These controls verify assay specificity and help distinguish true signal from background noise.

No—an IgG control is not required for CUT&Tag-IT.

CUT&Tag uses a tethered pA-Tn5 enzyme that is highly specific for antibody-bound genomic regions. Internal testing by Active Motif showed that an IgG control does not add meaningful information beyond the standard negative control.

• IgG controls are not used in downstream analysis
• IgG controls are not equivalent to INPUT controls used in ChIP-Seq
• Optional only if required for internal validation

Yes—library quality and concentration should always be checked before sequencing.

• Size distribution: Analyze libraries on a TapeStation or Bioanalyzer. Most fragments should be < 500 bp.
• Library concentration: Quantify using a KAPA Library Quantification Kit.

These QC steps ensure high-quality sequencing data and reduce the risk of failed runs.

Active Motif provides a curated list of antibodies validated specifically for CUT&Tag-IT.

View the full list of CUT&Tag-IT–validated antibodies here: CUT&Tag-validated antibodies

These antibodies have been tested for performance, specificity, and reproducibility with the CUT&Tag-IT workflow.

Not always. CUT&Tag and ChIP-Seq use very different workflows and binding conditions.

An antibody that performs well in ChIP-Seq does not necessarily work in CUT&Tag-IT. For reliable results, we recommend:

• Using CUT&Tag-IT–validated Active Motif antibodies, or
• Independently validating a ChIP-grade or ChIP-validated antibody in the CUT&Tag-IT workflow.

Antibody compatibility is one of the most important factors for successful CUT&Tag experiments.

Yes. The CUT&Tag-IT^® Express Assay Kit is compatible with both monoclonal and polyclonal antibodies.

The assay supports antibodies raised in mouse or rabbit, which bind efficiently to the pA-Tn5 transposome used in the CUT&Tag-IT workflow.

Tagged proteins have not been formally validated with CUT&Tag-IT.

However, in principle, CUT&Tag-IT should work with tagged proteins if a high-quality antibody against the tag is used. Performance may vary, so validation with your specific tag and target is recommended before running large experiments.

Yes. The CUT&Tag-IT^® Express Assay Kit includes 4×4 unique dual indexes for multiplexing up to 16 samples.

To multiplex more than 16 samples (up to 96), use Active Motif’s Nextera™-Compatible CDI and UDI Primers (1-96).

These primers match Illumina Nextera index sets (N701–N704 and N501–N504) and are supplied at 25 µM for direct use. You may also combine other Nextera-compatible primers at the same concentration.

Yes, but with important limitations. CUT&Tag-IT libraries can be analyzed by qPCR, however the results may be unreliable.

Because Tn5 inserts sequencing adapters at random positions, many fragments will not contain both primer binding sites. As a result, gene-specific qPCR primers may fail to amplify all target fragments.

Implication: qPCR can underestimate library abundance or target enrichment and should not be used as a quantitative replacement for sequencing.

They are dual-indexed.

Each CUT&Tag-IT library contains two unique index sequences (i7 and i5), enabling accurate sample demultiplexing and reducing index hopping on Illumina platforms.

No. CUT&Tag-IT libraries do not include molecular identifiers (UMIs).

Duplicate reads should be handled using standard alignment and duplicate-removal tools rather than UMI-based collapsing.

Paired-end sequencing is recommended.

Paired-end reads improve fragment mapping accuracy, peak resolution, and alignment confidence for CUT&Tag data.

We recommend paired-end 2 × 38 bp (PE38).

Although this is shorter than the read length reported in the original Henikoff CUT&Tag paper, Active Motif has not observed reduced mapping rates or data quality with PE38.

Longer read lengths may also be used if desired.

CUT&Tag data is analyzed using standard ChIP-Seq bioinformatics workflows.

Active Motif recommends aligning reads with BWA and performing peak calling with MACS2.

Downstream analysis (signal tracks, peak annotation, and differential binding) can be performed using the same tools and pipelines commonly used for ChIP-Seq datasets.